Gene transduction, the introduction of foreign genetic materials into cells or organisms, is a requisite technology involved in approaches to correcting genetic abnormalities, i.e., gene therapy. Genes can be transfected into cells by physical means such as scrape loading or ballistic penetration; by chemical means such as co-precipitation of DNA with calcium phosphate or liposomal encapsulation; or by electrophysiological means such as electroporation. However, these methods are relatively inefficient and the cells are significantly perturbed from their normal environment. In contrast, transduction of genes by means of recombinant viruses into a cell that is held in a physiologic environment takes advantage of the relative efficiency of viral infection processes.
Current gene therapy involves infection of organisms or cells with replication-deficient recombinant viruses containing the desired gene or genes. These viruses introduce the desired gene or genes into target cells by relatively efficient infection processes. However, these viruses are rendered deficient in some later replication step so that the primary infection does not produce progeny virus thereby circumventing the problem of propagating a deleterious full lytic viral infection cycle. Ideally, this virus only infects target cells, functionally expresses the gene it carries, and perpetuates the expression of this gene in the target cell and its progeny. Practically, however, this objective has been difficult to achieve. The reasons for these difficulties lie in the nature of the viral agents used to introduce the foreign gene(s) in question.
There are a number of replication-deficient viruses which are currently being used or have been proposed as gene transduction vectors. Examples include retroviruses, adenoviruses, adeno-associated viruses and herpesviruses. Although it has been demonstrated that gene therapy is possible using such viruses, all involve significant problems that limit or preclude their applicability to gene therapy in a clinically relevant setting.
Retroviruses such as MuLv may integrate into host cellular genomes, but these viral vectors only infect dividing cells. Thus, while they may be highly effective as transduction vectors, they cannot be used to stimulate gene expression in resting cells. This is a significant limitation for their utility in the treatment of many genetic disorders. Another limitation is that retroviruses become inactive at high concentrations and on exposure to human blood. Therefore, retroviruses cannot be concentrated in high enough amounts to avoid administering enormous volumes of fluid containing the recombinant viruses.
Adenovirus DNA does not integrate into the host genome, so when an infected cell divides, the viral DNA which is not replicated is then present in one-half of daughter cells. A second division dilutes still further the transduced gene until it is eventually lost to the cell progeny. Therefore, adenovirus mediated gene transduction allows expression in resting cells but does not permit transmission of gene expression to daughter cells. A further significant limitation is that adenovirus elicits a destructive immune response on the part of the host immune system that leads to the elimination of the desired transduced cells, much as the body would clear a clinical virus infection (e.g., influenza, rhinovirus).
Adeno-associated virus (AAV), but probably not recombinant AAV, integrates into the host genome. The wild-type AAV has a preference for an integration site that is at or near an important gene translocation site for some acute leukemias. Furthermore, like retroviruses, adeno-associated virus can not be produced at high virus concentrations.
Herpesviruses have only recently been proposed as vectors for gene transduction and their use has not been fully evaluated at this time. However, they appear to be most effective in the central nervous system and are not clearly useful in other organ targets.
SV40 (Simian Virus-40) has been demonstrated to provide a unique vector for gene therapy which has several advantages over any of the currently available viral vectors. It has now been found that integration of this vector into a selected site of the genome of a cell can be directed by flanking a nucleic acid sequence to be integrated with integration promotion sequences designed to target integration of the vector to the selected site of the cell's genome.